Renin-producing Cells Research Articles

Abstract MP14: Identifying markers of Concentric Arterial and Arteriolar Hypertrophy (CAAH) Using Multiomics.

Experimental or spontaneous mutations of any of the renin-angiotensin system (RAS) genes or treatment with RAS inhibitors in mammals lead to kidney concentric arterial and arteriolar hypertrophy (CAAH). Despite the prevalent use of RAS inhibitors in children and adults, our knowledge of this disease is very limited. We assessed the hypothesis that mouse models of CAAH will produce identifiable and reproducible markers of early and progressive disease. We generated murine models that tracks the lineage of renin-producing cells with deletion ( Ren1 c-/- ;Ren1 cCre ;R26R mTmG , Ren1cKO) or an intact renin gene ( Ren1 c+/- ;Ren1 cCre ;R26R mTmG , control). Kidney cells expressing GFP and tdTomato were isolated by FACS and processed for scRNA-seq. Plasma and urine were collected for proteomics, metabolomics and lipidomics. To study kidney function and structure, we measured transdermal glomerular filtration rate (tGFR) and performed comprehensive three-dimensional (CUBIC) and immunohistochemical analyses. Ren1cKO mice presented with a decrease in the GFR (1.47±0.02 vs 0.40±0.06 mL/min/100g b.w ( n= 6; P <0.05)). They also had polydipsia (3.9±0.05 vs 9.6±0.09 mL/24h ( n= 6; P <0.05), polyuria (2.4±0.07 vs 5.9±0.09 mL/24h ( n= 6; P <0.05) and marked reduction in urinary osmolality (1384±92.09 vs 588±68.04 mOsm/kg ( n= 6; P <0.05). The wall thickness of the afferent arterioles in Ren1cKO mice was significantly increased when compared to controls (4.54 ± 0.17 μm vs 11.57 ± 0.91 μm, P <0.05). The hypertrophic arterioles were covered along their lengths with cells positive for renin. Plasma metabolites showed a significant increase in fatty acids and phosphatidylcholines ( P <0.05) in Ren1cKO mice. Collagens ( P <0.05), lymphocyte antigens ( P <0.05), Spink 1 ( P <0.05), and cadherin 13 were differentially abundant with cadherin 13 exclusively detected in Ren1cKO mice at 1 and 6 months. Our scRNA-seq data also found Spink 1 ( P Holm-adj .=0.00) and Cadherin 13 ( P Holm-adj .=5.49X10 3 ) significantly increased in renin null cells, corroborating the proteomic findings. 1) CAAH involves smooth muscle cell transdifferentiation to renin-expressing cells that exhibit a mesenchymal phenotype characterized by pro-inflammatory pathways, and deregulation of lipid-metabolic homeostasis. Further, the exclusive expression of Spink and Cadherin 13 suggest their involvement in the pathogenesis of these severe, and silent arteriolar disease. Those molecules may serve to mark the initiation and progression of CAAH.

Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

Abstract P216: Multiomics Approach To Unravel The Molecular Mechanisms Responsible For Kidney Concentric Arterial And Arteriolar Hypertrophy

Introduction: Experimental or spontaneous mutations of any of the renin-angiotensin system (RAS) genes or treatment with RAS inhibitors in mammals -including humans- lead to kidney concentric arterial and arteriolar hypertrophy (CAAH), a silent, progressive, and severe thickening of the intrarenal arterial tree. Therefore, it is crucial to comprehend the identity and fate changes of the vascular smooth muscle cells (VSMCs) as they switch their phenotype to renin-producing cells (RPC) during the progression of the arteriolar disease. Objective: Using new mouse models and single-cell ‘omic’ approaches available in our lab to assess the chromatin landscape of the RPCs and VSMCs, we aim to identify the genomic hotspots that determine the initiation, evolution, and ultimate fate of the diseased cells. Methods: We generated two murine models. In the first model ( Ren1 c-/- ;Ren1 cCre ;R26R mTmG ) the renin gene is deleted while the cells (renin null cells) are labeled green (GFP+) from embryonic life. In the second model ( SMMHC CreERt2 ; R26R tdTomato ; Ren1 cYFP ) VSMCs are labeled red (tdTomato+) upon tamoxifen injection whereas cells currently expressing renin are labeled yellow (YFP+). Kidney cells expressing the different fluorophores were isolated by Fluorescence Activated Cell Sorting and processed for Single Cell Multiome ATAC (Assay for Transposase-Accessible Chromatin) + Gene Expression, using 10X Genomics Chromium technology. Results: The wall thickness of the afferent arterioles in Ren1 C KO mice was significantly increased when compared to control (4.54 ± 0.17 μm vs 11.57 ± 0.91 μm, P <0.0001). The hypertrophic arterioles were covered along their lengths with cells positive for renin, suggesting that renin cells were involved in the hypertrophy. As the mice got older, SMCs accumulate inwardly and concentrically, obstructing the vessels lumens, leading to focal fibrosis. Conclusion: Our studies showed that deletion of the renin gene led to CAAH. Experiments involving transcriptomics and epigenomics are ongoing to understand the evolution of the CAAH and identify and transcriptional profile changes that occur at the single-cell level.

Genome-Wide Association Studies for Albuminuria of Nondiabetic Taiwanese Population

Introduction: Chronic kidney disease, which is defined by a reduced estimated glomerular filtration rate and albuminuria, imposes a large health burden worldwide. Ethnicity-specific associations are frequently observed in genome-wide association studies (GWAS). This study conducts a GWAS of albuminuria in the nondiabetic population of Taiwan. Methods: Nondiabetic individuals aged 30–70 years without a history of cancer were enrolled from the Taiwan Biobank. A total of 6,768 subjects were subjected to a spot urine examination. After quality control using PLINK and imputation using SHAPEIT and IMPUTE2, a total of 3,638,350 single-nucleotide polymorphisms (SNPs) remained for testing. SNPs with a minor allele frequency of less than 0.1% were excluded. Linear regression was used to determine the relationship between SNPs and log urine albumin-to-creatinine ratio. Results: Six suggestive loci are identified in or near the FCRL3 (p = 2.56 × 10⁻⁶), TMEM161 (p = 4.43 × 10⁻⁶), EFCAB1 (p = 2.03 × 10⁻⁶), ELMOD1 (p = 2.97 × 10⁻⁶), RYR3 (p = 1.34 × 10⁻⁶), and PIEZO2 (p = 2.19 × 10⁻⁷). Genetic variants in the FCRL3 gene that encode a secretory IgA receptor are found to be associated with IgA nephropathy, which can manifest as proteinuria. The PIEZO2 gene encodes a sensor for mechanical forces in mesangial cells and renin-producing cells. Five SNPs with a p-value between 5 × 10⁻⁶ and 5 × 10⁻⁵ are also identified in five genes that may have a biological role in the development of albuminuria. Conclusion: Five new loci and one known suggestive locus for albuminuria are identified in the nondiabetic Taiwanese population.

Cells of the renin lineage promote kidney regeneration post-release of ureteral obstruction in neonatal mice.

Ureteral obstruction leads to significant changes in kidney renin expression. It is unclear whether those changes are responsible for the progression of kidney damage, repair, or regeneration. In the current study, we aimed to elucidate the contribution of renin-producing cells (RPCs) and the cells of the renin lineage (CoRL) towards kidney damage and regeneration using a model of partial and reversible unilateral ureteral obstruction (pUUO) in neonatal mice. Renin cells are progenitors for other renal cell types collectively called CoRL. We labeled the CoRL with green fluorescent protein (GFP) using genetic approaches. We performed lineage tracing to analyze the changes in the distribution of CoRL during and after the release of obstruction. We also ablated the RPCs and CoRL by cell-specific expression of Diphtheria Toxin Sub-unit A (DTA). Finally, we evaluated the kidney damage and regeneration during and after the release of obstruction in the absence of CoRL. In the obstructed kidneys, there was a 163% increase in the renin-positive area and a remarkable increase in the distribution of GFP+ CoRL. Relief of obstruction abrogated these changes. In addition, DTA-expressing animals did not respond to pUUO with increased RPCs and CoRL. Moreover, reduction in CoRL significantly compromised the kidney's ability to recover from the damage after the release of obstruction. CoRL play a role in the regeneration of the kidneys post-relief of obstruction.

Upregulation of Piezo2 in the mesangial, renin, and perivascular mesenchymal cells of the kidney of Dahl salt-sensitive hypertensive rats and its reversal by esaxerenone.

The recent discovery of mechanosensitive ion channels has promoted mechanobiological research in the field of hypertension and nephrology. We previously reported Piezo2 expression in mouse mesangial and juxtaglomerular renin-producing cells, and its modulation by dehydration. This study aimed to investigate how Piezo2 expression is altered in hypertensive nephropathy. The effects of the nonsteroidal mineralocorticoid receptor blocker, esaxerenone, were also analyzed. Four-week-old Dahl salt-sensitive rats were randomly assigned to three groups: rats fed a 0.3% NaCl diet (DSN), rats fed a high 8% NaCl diet (DSH), and rats fed a high salt diet supplemented with esaxerenone (DSH + E). After six weeks, DSH rats developed hypertension, albuminuria, glomerular and vascular injuries, and perivascular fibrosis. Esaxerenone effectively decreased blood pressure and ameliorated renal damage. In DSN rats, Piezo2 was expressed in Pdgfrb-positive mesangial and Ren1-positive cells. Piezo2 expression in these cells was enhanced in DSH rats. Moreover, Piezo2-positive cells accumulated in the adventitial layer of intrarenal small arteries and arterioles in DSH rats. These cells were positive for Pdgfrb, Col1a1, and Col3a1, but negative for Acta2 (αSMA), indicating that they were perivascular mesenchymal cells different from myofibroblasts. Piezo2 upregulation was reversed by esaxerenone treatment. Furthermore, Piezo2 inhibition by siRNA in the cultured mesangial cells resulted in upregulation of Tgfb1 expression. Cyclic stretch also upregulated Tgfb1 in both transfections of control siRNA and Piezo2 siRNA. Our findings suggest that Piezo2 may have a contributory role in modulating the pathogenesis of hypertensive nephrosclerosis and have also highlighted the therapeutic effects of esaxerenone on salt-induced hypertensive nephropathy. Mechanochannel Piezo2 is known to be expressed in the mouse mesangial cells and juxtaglomerular renin-producing cells, and this was confirmed in normotensive Dahl-S rats. In salt-induced hypertensive Dahl-S rats, Piezo2 upregulation was observed in the mesangial cells, renin cells, and notably, perivascular mesenchymal cells, suggesting its involvement in kidney fibrosis.

A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension.

Genetic variants in the third intron of the PRDM6 gene have been associated with BP traits in multiple GWAS. By combining fine mapping, massively parallel reporter assays, and gene editing, we identified super enhancers that drive the expression of PRDM6 and are partly regulated by STAT1 as the causal variants for hypertension. The heterozygous disruption of Prdm6 in mice expressing Cre recombinase under the control of mouse smooth muscle cell protein 22-α promoter (Prdm6fl/+ SM22-Cre) exhibited a markedly higher number of renin-producing cells in the kidneys at E18.5 compared with WT littermates and developed salt-induced systemic hypertension that was completely responsive to the renin inhibitor aliskiren. Strikingly, RNA-Seq analysis of the mouse aortas identified a network of PRDM6-regulated genes that are located in GWAS-associated loci for blood pressure, most notably Sox6, which modulates renin expression in the kidney. Accordingly, the smooth muscle cell-specific disruption of Sox6 in Prdm6fl/+ SM22-Cre mice resulted in a dramatic reduction of renin. Fate mapping and histological studies also showed increased numbers of neural crest-derived cells accompanied by increased collagen deposition in the kidneys of Prdm6fl/+ Wnt1Cre-ZsGreen1Cre mice compared with WT mice. These findings establish the role of PRDM6 as a regulator of renin-producing cell differentiation into smooth muscle cells and as an attractive target for the development of antihypertensive drugs.

PS-BPB06-2: PIEZO2 EXPRESSION AND ITS ALTERATION BY MECHANICAL FORCES IN MOUSE MESANGIAL CELLS AND RENIN PRODUCING CELLS

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1 positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin producing cells, along with the overproduction of renin and enlargement of the area of renin producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.

Abstract 090: Cells Of The Renin Lineage Promote Kidney Regeneration Post - Release Of Ureteral Obstruction In Neonatal Mice.

Renin, the rate-limiting enzyme of the renin-angiotensin system, is increased in the kidneys subjected to ureteral obstruction. We elucidated the changes and contribution of renin-producing cells (RPCs) and the cells of renin lineage (CoRL) during kidney damage and regeneration using a partial unilateral ureteral obstruction (pUUO) model in neonatal mice. Renin cells were ablated by cell-specific expression of diphtheria toxin A chain (DTA) and labeled with GFP. We compared the cell fate changes between kidneys from the wild-type (WT) and the DTA-expressing animals, using the pUUO model. The kidneys of 3W old DTA-positive Ren1 dDTA/+ ;R26R DTA/+ and Ren1 dCre/+ ;R26R DTA/+ genotypes were grossly normal. We observed no significant differences in the Myh11-positive renal vascular area between the WT and the DTA-positive mice (WT-3W: 1.72±0.10 % (n=6), Ren1 dDTA/+ ;R26R DTA/+ -3W: 1.94 ±0.123 % (n=7), Ren1 dCre/+ ;R26R DTA/+ -3W: 2.017±0.16% (n=4)). In the kidneys of the WT group, RPCs and the CoRL significantly increased at 3W post-obstruction and the distribution was similar to the sham-operated kidneys at 2W post-relief of obstruction (Renin-positive area-WT: Sham-3W: 0.57±0.02% (n=6), Obstructed-3W: 1.59±0.44% (n=4), Post-release-2W: 0.71±0.12% (n=3)). There was a significant decrease in RPCs in the DTA animals, and the reduction of RPCs and CoRL was markedly higher in Ren1 dCre/+ ;R26R DTA/+ mice in comparison to Ren1 dDTA/+ ;R26R DTA/+ group. Also, the DTA-positive animals did not respond to pUUO with a significant increase in renin as in the WT (Renin-positive area: Ren1 dDTA/+ ;R26R DTA/+ Sham-3W: 0.13±0.048% (n=5), Obstructed-3W: 0.23±0.10% (n=5), Post-release-2W: 0.14±0.04% (n=9) Ren1 dCre/+ ;R26R DTA/+ Sham: 0.010±0.004% (n=4), Obstructed: 0.07±0.004% (n=4), Post-release: 0.03±0.004% (n=3)). The renal fibrotic area and Tgfb1 mRNA levels post-obstruction and relief of obstruction were significantly high in Ren1 dCre/+ ;R26R DTA/+ group in comparison to Ren1 dDTA/+ ;R26R DTA/+ genotype due to the depletion of CoRL. Thus, our current study reveals that CoRL undergoes cell fate changes in response to obstruction and the release of obstruction, and these changes play a critical role in promoting kidney regeneration post-relief of obstruction.

FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses

Abstract BACKGROUND AND AIMS The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma. METHOD We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly. RESULTS In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection. CONCLUSION The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.

Expression of Pannexin 1 in the Human Kidney during Embryonal, Early Fetal and Postnatal Development and Its Prognostic Significance in Diabetic Nephropathy

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman’s capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

Piezo2 expression and its alteration by mechanical forces in mouse mesangial cells and renin-producing cells

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.

The endocrine kidney: tampering with oxygen sensors may change your character

The endocrine kidney: tampering with oxygen sensors may change your character

Prolyl-4-hydroxylases 2 and 3 control erythropoietin production in renin-expressing cells of mouse kidneys.

Activation of the hypoxia-signalling pathway induced by deletion of the ubiquitin-ligase von Hippel-Lindau protein causes an endocrine shift of renin-producing cells to erythropoietin (EPO)-expressing cells. However, the underlying mechanisms have not yet been investigated. Since oxygen-regulated stability of hypoxia-inducible transcription factors relevant for EPO expression is dependent on the activity of prolyl-4-hydroxylases (PHD) 2 and 3, this study aimed to determine the relevance of different PHD isoforms for the EPO expression in renin-producing cells in vivo. For this purpose, mice with inducible renin cell-specific deletions of different PHD isoforms were analysed. Our study shows that there are two subgroups of renal renin-expressing cells, juxtaglomerular renin+ cells and platelet-derived growth factor receptor-β+ interstitial renin+ cells. These interstitial renin+ cells belong to the cell pool of native EPO-producing cells and are able to express EPO and renin in parallel. In contrast, co-deletion of PHD2 and PHD3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular and hyperplastic renin+ cells and downregulates renin expression. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent the hypoxia-inducible transcription factor-2-dependent phenotype shift into EPO cells. In summary, PHDs seem important for the stabilization of the juxtaglomerular renin cell phenotype. Moreover, these findings reveal tubulointerstitial cells as a novel site of renal renin expression and suggest a high endocrine plasticity of these cells. Our data concerning the distinct expression patterns and functions of PHD2 and PHD3 provide new insights into the regulation of renin-producing cells and highlight the need for selective PHD inhibitors. KEY POINTS: Renal renin-expressing cells can be clearly distinguished into two subgroups, the typical juxtaglomerular renin-producing cells and interstitial renin+ cells. Interstitial renin+ cells belong to the cell pool of native erythropoietin (EPO)-producing cells, show a fast EPO response to acute hypoxia-inducible factor-2 (HIF-2) stabilization and are able to express EPO and renin in parallel. Only co-deletion of the prolyl-4-hydroxylases (PHD) 2 and 3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular renin+ cells. Chronic HIF-2 stabilization in juxtaglomerular renin-expressing cells leads to their phenotypic shift into EPO-producing cells. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent a HIF-2-dependent phenotype shift into EPO cells suggesting PHD3 fulfils a stabilizer function for the juxtaglomerular renin cell phenotype.

Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203.

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), and it is a known risk factor for hypertension. The upregulation of the renin-angiotensin system has been reported in IH, and the correlation between renin and CD38 has been noted. We exposed human HEK293 and mouse As4.1 renal cells to experimental IH or normoxia for 24 h and then measured the mRNA levels using a real-time reverse transcription polymerase chain reaction. The mRNA levels of Renin (Ren) and Cd38 were significantly increased by IH, indicating that they could be involved in the CD38-cyclic ADP-ribose signaling pathway. We next investigated the promotor activities of both genes, which were not increased by IH. Yet, a target mRNA search of the microRNA (miRNA) revealed both mRNAs to have a potential target sequence for miR-203. The miR-203 level of the IH-treated cells was significantly decreased when compared with the normoxia-treated cells. The IH-induced upregulation of the genes was abolished by the introduction of the miR-203 mimic, but not the miR-203 mimic NC negative control. These results indicate that IH stress downregulates the miR-203 in renin-producing cells, thereby resulting in increased mRNA levels of Ren and Cd38, which leads to hypertension.

Patterns of differentiation of renin lineage cells during nephrogenesis.

Developmentally heterogeneous renin-expressing cells serve as progenitors for mural, glomerular, and tubular cells during nephrogenesis and are collectively termed renin lineage cells (RLCs). In this study, we quantified different renal vascular and tubular cell types based on specific markers and assessed proliferation and de novo differentiation in the RLC population. We used kidney sections of mRenCre-mT/mG mice throughout nephrogenesis. Marker positivity was evaluated in whole digitalized sections. At embryonic day 16, RLCs appeared in the developing kidney, and the expression of all stained markers in RLCs was observed. The proliferation rate of RLCs did not differ from the proliferation rate of non-RLCs. RLCs expanded mainly by de novo differentiation (neogenesis). Fractions of RLCs originating from the stromal progenitors of the metanephric mesenchyme (renin-producing cells, vascular smooth muscle cells, and mesangial cells) decreased during nephrogenesis. In contrast, aquaporin-2-positive RLCs in the collecting duct system, which embryonically emerges almost exclusively from the ureteric bud, expanded postpartum. The cubilin-positive RLC fraction in the proximal tubule, deriving from the cap mesenchyme, remained constant. In summary, RLCs were continuously detectable in the vascular and tubular compartments of the kidney during nephrogenesis. Therein, various patterns of RLC differentiation that depend on the embryonic origin of the cells were identified.NEW & NOTEWORTHY The unifying feature of the renal renin lineage cells (RLCs) is their origin from renin-expressing progenitors. RLCs evolve to an embryologically heterogeneous large population in structures with different ancestry. RLCs are also targets for the widely used renin-angiotensin-system blockers, which modulate their phenotype. Unveiling the different differentiation patterns of RLCs in the developing kidney contributes to understanding changes in their cell fate in response to homeostatic challenges and the use of antihypertensive drugs.

A Compendium on Hypertension: New Advances and Future Impact.

A Compendium on Hypertension: New Advances and Future Impact.

Localization of angiotensin II type 1 receptor gene expression in rodent and human kidneys.

The kidneys are an important target for angiotensin II (ANG II). In adult kidneys, the effects of ANG II are mediated mainly by ANG II type 1 (AT1) receptors. AT1 receptor expression has been reported for a variety of different cell types within the kidneys, suggesting a broad spectrum of actions for ANG II. Since there have been heterogeneous results in the literature regarding the intrarenal distribution of AT1 receptors, this study aimed to obtain a comprehensive overview about the localization of AT1 receptor expression in mouse, rat, and human kidneys. Using the cell-specific and high-resolution RNAscope technique, we performed colocalization experiments with various cell markers to specifically discriminate between different segments of the tubular and vascular system. Overall, we found a similar pattern of AT1 mRNA expression in mouse, rat, and human kidneys. AT1 receptors were detected in mesangial cells and renin-producing cells. In addition, AT1 mRNA was found in interstitial cells of the cortex and outer medulla. In rodents, late afferent and early efferent arterioles expressed AT1 receptor mRNA, but larger vessels of the investigated species showed no AT1 expression. Tubular expression of AT1 mRNA was species dependent with a strong expression in proximal tubules of mice, whereas expression was undetectable in human tubular cells. These findings suggest that the (juxta)glomerular area and tubulointerstitium are conserved expression sites for AT1 receptors across species and might present the main target sites for ANG II in adult human and rodent kidneys.NEW & NOTEWORTHY Angiotensin II (ANG II) type 1 (AT1) receptors are essential for mediating the effects of ANG II in the kidneys. This study aimed to obtain a comprehensive overview about the cell-specific localization of AT1 receptor expression in rodent and human kidneys using the novel RNAscope technique. We found that the conserved AT1 receptor mRNA expression sites across species are the (juxta)glomerular areas and tubulointerstitium, which might present main target sites for ANG II in adult human and rodent kidneys.

Role of Pericytes in the Development of the Renin/Angiotensin System: Induction of Functional Renin in Cultures of Pericytes.

Renal pericytes have a critical importance for angiogenesis and vascular remodeling, medullary blood flow regulation, and development of fibrosis. An emerging role for kidney pericytes is their ability to induce renin expression and synthesis. Here, we present methods for purification of human renal pericytes, their primary culture, and differentiation into renin-producing cells. Possible applications of these protocols include investigations into (1) renin cell recruitment mechanisms, (2) modulation of renin expression/secretion by small molecules, and (3) renin expression/secretion in nonrenal pericytes. A potential therapeutic application of this work is the identification of new players regulating the renin-angiotensin system.

Functional Relevancies of Trans-Differentiation in the Juxtaglomerular Apparatus of Rat Kidney.

Glomerular filtration rate is controlled by the contractile effect of angiotensin II on afferent and efferent arterioles. The renin positivity of the afferent arterioles depends on tubuloglomerular feedback via the macula densa (MD) and short loop feedback via the afferent arteriolar endothelia. The renin-producing cells are trans-differentiated from smooth muscle cells (SMCs) of mainly the afferent arterioles, the MD cells are trans-differentiated from the neighboring tubular cells, and the high-permeability endothelial cells are trans-differentiated from normal permeability endothelial cells facing the renin-negative part of the afferent arterioles. All of the trans-differentiations depend on the activity of the renin-angiotensin system (RAS). The distribution of AT1 receptors for angiotensin II expresses the contractile effects of angiotensin II on renin-negative SMCs and the negative effect on trans-differentiation of renin-positive SMCs and MD cells. The purpose of this review is to summarize the stereological data of molecules like angiotensin II AT1 receptors, L-type calcium channels, and renin receptors in the juxtaglomerular apparatus of normal and STZ-induced diabetic rat kidneys, thus showing their functional relevancies on trans-differentiation among the juxtaglomerular apparatus’ elements.